Testis tissue xenografting (TTX) provides a novel in vivo model for the study of testis function, and a previously-unavailable opportunity to produce spermatozoa in the grafts from immature donors of diverse species. The overall objectives of this thesis were to examine a number of factors that potentially affect the outcome of TTX, and to apply TTX using immature bison and deer donors as models for endangered ungulates. The objective of the first experiment was to examine the effects of recipient mouse strain, gender and gonadal status on the outcome of TTX. Eight small fragments of neonatal porcine testis tissue (~5 mg each) were grafted under the back skin of immunodeficient mice of different strains (SCID vs. nude), gender (male vs. female), and gonadal status (intact vs. gonadectomised), using a 2×2×2 factorial design (8 groups, n = 7 mice/group). The xenografts were recovered at 8 mo post-grafting and evaluated for gross and histological attributes. Gonadectomy of the recipients did not affect any of the measured outcomes of TTX (P > 0.05), and data were pooled into four groups based on recipient strain and gender. Overall, male recipient mice had grafts with higher mean (+SEM) recovery rate (97 ± 2.3% vs. 88 ± 2.4%, P = 0.004), weight (348 ± 26.3 vs. 104 ± 27.0 mg, P < 0.001), seminiferous tubular diameter (150 ± 3.3 vs. 108 ± 5.3 mg, P < 0.001), percentage of tubules containing spermatozoa (32 ± 3.2 vs. 6 ± 1.8%, P < 0.001), elongated spermatids (13 ± 1.4% vs. 4 ± 0.8%, P < 0.001), and round spermatids (10 ± 1.2% vs. 6 ± 1.1%, P = 0.006) than female mice. Overall, SCID mice had grafts with higher recovery rate (98 ± 2.4% vs. 87 ± 2.3%, P = 0.001), average weight (292 ± 27.0 vs. 160 ± 26.3 mg, P = 0.001), tubular density (44 ± 3.3 vs. 33 ± 2.1, P = 0.02), percentage of tubular cross-sections containing spermatocytes (27 ± 3.7% vs. 13 ± 2.3%, P = 0.003) than nude mice. Among the four groups of recipients, the grafts from male SCID mice had the highest weight (P < 0.05) and percentage of tubules containing spermatozoa (P < 0.05).<p> The objective of the second experiment was to evaluate the effect of using different numbers of donor testis tissue fragments on the outcome of TTX. Fragments of donor piglet testis tissue were grafted subcutaneously under the back skin of four groups of castrated male nude mice (n = 10/group). Each group of recipient mice received 2, 4, 8, or 16 fragments per mouse. Mice were sacrificed at 8 mo post-grafting, and xenografts were evaluated for physical growth and histological development. The relative weight of the vesicular gland (index) was also determined as a measure of bioactive androgen production by grafts in castrated recipient mice. The overall graft recovery rate was ~94% (range 86-98%) which did not differ among the groups (P > 0.05). The group of mice that received 16 testis tissue fragments had higher mean (+ SEM) graft weights (278 ± 39.4 vs. 106 ± 38.0, P = 0.02), total graft weight (2,443 ± 338.8 vs. 192 ± 76.2, P < 0.001), vesicular gland index (0.5 ± 0.06 vs. 0.1 ± 0.06, P = 0.007), and percentage of seminiferous tubules with round spermatids (11 ± 1.5 vs. 3 ± 1.3, P = 0.03) than the group of mice that received two testis tissue fragments. The objective of the third experiment was to assess the use to salvage testis tissue from neonatal/immature bison or deer donors using TTX into immunodeficient recipient mice as models for closely-related rare or endangered ungulates. Donor testis tissue fragments from two newborn bison calves (Bison bison bison) and a 2-mo-old white-tailed deer fawn (Odocoileus virginianus) were grafted under the back skin of gonadectomised nude mice (n = 15 and n = 7 for bison and deer groups, respectively, 8 testis fragments/mouse). To examine the potential effect of individual donors, we grafted four testis tissue fragments from one bison calf on one side of the recipient and four fragments from the second bison calf on the other side. Single grafts were surgically removed from representative recipient mice every 2 mo for up to 16- and 14 mo post-grafting, for bison and deer groups, respectively. The overall graft recovery rates were 69% and 63% for bison and deer groups, respectively. For bison grafts, a donor effect on efficiency of spermatogenesis was also observed. The weight of bison testis tissue xenografts increased (P < 0.02) ~4-fold by 2 mo and ~10-fold by 16 mo post-grafting, and gradual maturational changes were evident in the form of seminiferous tubule expansion starting at 2 mo, first appearance of spermatocytes at 6 mo, round spermatids at 12 mo, and elongated spermatids at 16 mo post-grafting. Testis tissue xenografts from donor white-tailed deer also showed a gradual development starting with tubular expansion by 2 mo and presence of spermatocytes by 6 mo post-grafting, round and elongated spermatids by 8 mo, followed by fully-formed spermatozoa by 12 mo post-grafting. The timing of complete spermatogenesis roughly corresponded to the reported timing of sexual maturation in these species.<p> Taken together, the findings in this thesis suggest that male SCID mice provide a more suitable recipient model for TTX with neonatal porcine testis tissue; recipient mice can be grafted with as many as 16 testis tissue fragments for optimal results; and that TTX is a feasible strategy for salvaging genetic materials from immature males of rare or endangered ungulates that die prematurely.

Testis tissue xenografting (TTX) provides a novel in vivo model for the study of testis function, and a previously-unavailable opportunity to produce spermatozoa in the grafts from immature donors of diverse species. The overall objectives of this thesis were to examine a number of factors that potentially affect the outcome of TTX, and to apply TTX using immature bison and deer donors as models for endangered ungulates. The objective of the first experiment was to examine the effects of recipient mouse strain, gender and gonadal status on the outcome of TTX. Eight small fragments of neonatal porcine testis tissue (~5 mg each) were grafted under the back skin of immunodeficient mice of different strains (SCID vs. nude), gender (male vs. female), and gonadal status (intact vs. gonadectomised), using a 2×2×2 factorial design (8 groups, n = 7 mice/group). The xenografts were recovered at 8 mo post-grafting and evaluated for gross and histological attributes. Gonadectomy of the recipients did not affect any of the measured outcomes of TTX (P > 0.05), and data were pooled into four groups based on recipient strain and gender. Overall, male recipient mice had grafts with higher mean (+SEM) recovery rate (97 ± 2.3% vs. 88 ± 2.4%, P = 0.004), weight (348 ± 26.3 vs. 104 ± 27.0 mg, P < 0.001), seminiferous tubular diameter (150 ± 3.3 vs. 108 ± 5.3 mg, P < 0.001), percentage of tubules containing spermatozoa (32 ± 3.2 vs. 6 ± 1.8%, P < 0.001), elongated spermatids (13 ± 1.4% vs. 4 ± 0.8%, P < 0.001), and round spermatids (10 ± 1.2% vs. 6 ± 1.1%, P = 0.006) than female mice. Overall, SCID mice had grafts with higher recovery rate (98 ± 2.4% vs. 87 ± 2.3%, P = 0.001), average weight (292 ± 27.0 vs. 160 ± 26.3 mg, P = 0.001), tubular density (44 ± 3.3 vs. 33 ± 2.1, P = 0.02), percentage of tubular cross-sections containing spermatocytes (27 ± 3.7% vs. 13 ± 2.3%, P = 0.003) than nude mice. Among the four groups of recipients, the grafts from male SCID mice had the highest weight (P < 0.05) and percentage of tubules containing spermatozoa (P < 0.05).<p> The objective of the second experiment was to evaluate the effect of using different numbers of donor testis tissue fragments on the outcome of TTX. Fragments of donor piglet testis tissue were grafted subcutaneously under the back skin of four groups of castrated male nude mice (n = 10/group). Each group of recipient mice received 2, 4, 8, or 16 fragments per mouse. Mice were sacrificed at 8 mo post-grafting, and xenografts were evaluated for physical growth and histological development. The relative weight of the vesicular gland (index) was also determined as a measure of bioactive androgen production by grafts in castrated recipient mice. The overall graft recovery rate was ~94% (range 86-98%) which did not differ among the groups (P > 0.05). The group of mice that received 16 testis tissue fragments had higher mean (+ SEM) graft weights (278 ± 39.4 vs. 106 ± 38.0, P = 0.02), total graft weight (2,443 ± 338.8 vs. 192 ± 76.2, P < 0.001), vesicular gland index (0.5 ± 0.06 vs. 0.1 ± 0.06, P = 0.007), and percentage of seminiferous tubules with round spermatids (11 ± 1.5 vs. 3 ± 1.3, P = 0.03) than the group of mice that received two testis tissue fragments. The objective of the third experiment was to assess the use to salvage testis tissue from neonatal/immature bison or deer donors using TTX into immunodeficient recipient mice as models for closely-related rare or endangered ungulates. Donor testis tissue fragments from two newborn bison calves (Bison bison bison) and a 2-mo-old white-tailed deer fawn (Odocoileus virginianus) were grafted under the back skin of gonadectomised nude mice (n = 15 and n = 7 for bison and deer groups, respectively, 8 testis fragments/mouse). To examine the potential effect of individual donors, we grafted four testis tissue fragments from one bison calf on one side of the recipient and four fragments from the second bison calf on the other side. Single grafts were surgically removed from representative recipient mice every 2 mo for up to 16- and 14 mo post-grafting, for bison and deer groups, respectively. The overall graft recovery rates were 69% and 63% for bison and deer groups, respectively. For bison grafts, a donor effect on efficiency of spermatogenesis was also observed. The weight of bison testis tissue xenografts increased (P < 0.02) ~4-fold by 2 mo and ~10-fold by 16 mo post-grafting, and gradual maturational changes were evident in the form of seminiferous tubule expansion starting at 2 mo, first appearance of spermatocytes at 6 mo, round spermatids at 12 mo, and elongated spermatids at 16 mo post-grafting. Testis tissue xenografts from donor white-tailed deer also showed a gradual development starting with tubular expansion by 2 mo and presence of spermatocytes by 6 mo post-grafting, round and elongated spermatids by 8 mo, followed by fully-formed spermatozoa by 12 mo post-grafting. The timing of complete spermatogenesis roughly corresponded to the reported timing of sexual maturation in these species.<p> Taken together, the findings in this thesis suggest that male SCID mice provide a more suitable recipient model for TTX with neonatal porcine testis tissue; recipient mice can be grafted with as many as 16 testis tissue fragments for optimal results; and that TTX is a feasible strategy for salvaging genetic materials from immature males of rare or endangered ungulates that die prematurely.

The nervous system and immune system contact one another through two-way communication in order to establish and preserve homeostasis. The sympathetic neurotransmitter norepinephrine has an impact on how the immune system responds by affecting regional blood flow and activation of adrenergic receptors on leukocytes. Former studies showed that immune cells are capable of releasing nerve growth factor allowing for the establishment and continuation of sympathetic nerves in targeted tissues. From this gathered information, it was hypothesized that sympathetic nerves would prove to be less frequent in spleens from the immunodeficient R2G2 mouse strain (Envigo) when compared to 129P3/J (129) and C57BL/6 (C57) strains. R2G2 mice are an immunodeficient strain that lacks functional T, B, and natural killer cells. Ten to eleven week aged-matched male mice were measured by body weight, spleen weight, and temperature. Spleens were cut and fixed for histological investigation. Sympathetic nerves were labeled by immunostaining tyrosine hydroxylase (TH). Hematoxylin & eosin (H&E) was used to stain spleen sections in order to evaluate cytoarchitecture. Von Willebrand factor (VWF) was used to immunostain for megakaryocytes. R2G2 mice showed slightly higher temperatures and body weights but yielded a significantly smaller spleen weight (R2G2, 38.20 ± 1.48; 129, 65.08 ± 11.71; C57, 81.33 ± 8.38; P< 0.0001, ANOVA). TH stain revealed sympathetic innervation in all strains but location and morphology differed in R2G2 mice compared to controls. Control spleens had nerves which entered white pulp regions of the spleen and were closely related to leukocytes. Fiber profiles in the controls were filamentous with small acute bends. R2G2 differed by having (TH+) nerve fibers more associated with arteries and less localized in the surrounding parenchyma. The fibers were abnormally swollen and held a more granular shape instead of a filamentous shape. The H&E stain showed clear red and white pulp zones in the control spleens with 129 showing more distinct germinal centers than C57. R2G2 H&E sections showed cytoarchitecture with indistinct pulp areas. VWF staining revealed R2G2 mice had an abundant amount of megakaryocytes versus control mice megakaryocyte counts (R2G2, 11.28 ± 3.87 per 20X field; 129, 1.73 ± 0.70; C57, 1.42 ± 0.13; P< 0.0001, ANOVA) and extramedullary hematopoiesis was highly prominent. This evidence supports that leukocytes secrete neurotrophic factors or are vital to establishing normal growth of TH+ nerves toward the white pulp. Leukocytes may not be required for sympathetic innervation of blood vessels in the spleen, however, lack of leukocytes shows TH+ nerve fibers with abnormal morphology in severely immune threatened mice.

O transplante de células-tronco hematopoéticas (CTHs) é o tipo de terapia celular mais usada atualmente. As células-tronco da medula óssea humana, do sangue periférico mobilizado e do sangue do cordão umbilical (SCU) são as únicas fontes de células utilizadas clinicamente para a recuperação hematopoética, mas elas têm uma disponibilidade limitada e apenas um terço dos pacientes apresentam um doador compatível. Uma fonte alternativa para suprir esta demanda seriam as células hematopoéticas derivadas a partir de células-tronco pluripotentes. Célulastronco embrionárias (CTE) são um tipo de células pulripotentes caracterizadas por sua capacidade ilimitada de autorrenovação e diferenciação em todas as células especializadas do indivíduo adulto. A alta capacidade de diferenciação dessas células em linhagens específicas por métodos de indução in vitro faz delas grandes promessas para o desenvolvimento de novas tecnologias aplicáveis à medicina regenerativa e terapia celular. Entretanto, ainda é necessário determinar se células-tronco hematopoéticas (CTH) geradas a partir de células pluripotentes são funcionais in vivo. Assim, o objetivo desse estudo consistiu no desenvolvimento de um modelo animal para o transplante de células hematopoéticas humanas provenientes de células pluripotentes e utilizamos CTH de sangue de cordão umbilical como controle. Para tanto, realizamos a padronização da dose de irradiação subletal nos camundongos NOD / SCID IL2R ? null (NSG) e utilizamos duas concentrações de células, 1x106 e 0,75x106 de células hematopoéticas diferenciadas a partir de CTE e células do cordão umbilical. Os animais que receberam as células do SCU apresentaram uma taxa mais elevada de enxertia em comparação aos que receberam ás células diferenciadas a partir de CTE humanas. Foi confirmado que após quinze dias do transplante, a hematopoese é restabelecida e que células CD45+ humanas estavam presentes, porém em baixas quantidades. Sobretudo, os resultados aqui apresentados enfatizaram o descrito na literatura, porém não ultrapassando a 1,5% de enxertia na medula óssea. Estes dados indicaram que os camundongos NSG proporcionaram um microambiente hematopoético favorável para as CTE humanas porém, essas células precisam ser investigadas no que tange aos fatores que aumentem de sua duração in vivo, otimizando a enxertia. / Hematopoietic stem cell transplantation (HSC) is the most widely used type of cell therapy nowadays. Human bone marrow, mobilized peripheral blood, and stem cells in the umbilical cord (UC) are the only sources of cells used clinically for hematopoietic recovery, but they have limited availability and only a third of patients have a matching donor. An alternative source to supply this demand would be hematopoietic cells derived from pluripotent stem cells. Embryonic stem cells (ESC) are a type of pulp-like cells characterized by their unlimited capacity for self-renewal and differentiation in all specialized cells of the adult individual. The high differentiation capacity of these cells in specific strains by in vitro induction methods makes great promises for the development of new technologies applicable to regenerative medicine and cell therapy. However, it remains to be determined whether hematopoietic stem cells (HTC) generated from pluripotent cells are functional in vivo. Thus, the objective of this study was to develop an animal model for the transplantation of human hematopoietic cells from pluripotent cells and to use HTC in the umbilical cord blood as a control. To do so, we performed standardization of the sublethal irradiation dose on the NOD / SCID IL2R ? null (NSG) mice and used two concentrations of cells, 1x106 and 0.75x106 hematopoietic cells differentiated from ESC and umbilical cord cells. The animals that received UC cells had a higher rate of grafting compared to those that received the differentiated cells from the human ESC. It was confirmed that after fifteen days of transplantation, hematopoiesis restored and that human CD45+ cells were present, but in low amounts. Above all, the results presented here emphasize the described in the literature, but not exceeding 1.5% of grafting in bone marrow. These data indicated that the NSG mice provided a hematopoietic microenvironment favorable to the human ESC, however, these cells need to be investigated with regard to factors that increase their duration in vivo, optimizing grafting.

The aim of this thesis was to study the mechanisms of innate imunity involved in the degradation of tumor cells on which the ligands of phagocytic receptors were installed. For this purpose both in vivo experiments using immunodeficient mice, and in vitro experiments based on monitoring the levels of inflammatory cytokines produced in the tumor tissue and on measuring the level of myeloperoxidase released during neutrophil degranulation were performed.

No description available.

No

Queratinócitos são células bastante atrativas para a transferência gênica ex vivo e liberação sistêmica, uma vez que as proteínas secretadas por estas células podem atingir a circulação via um mecanismo similar ao processo natural. No presente trabalho, queratinócitos transduzidos mediante um vetor retroviral com o gene do hormônio de crescimento de camundongo (mGH) foram submetidos a um tratamento de aderência ao colágeno e à análise clonal, com o intuito de enriquecer esta população de queratinócitos em células-tronco. O principal resultado foi um aumento da viabilidade celular in vitro dos queratinócitos tratados, que poderá se refletir num aumento da durabilidade da secreção do hormônio in vivo, quando realizado o implante de culturas organotípicas em camundongos anões imunodeficientes (lit/scid). Foi também utilizado um modelo de terapia gênica in vivo, baseado na eletrotransferência de DNA plasmidial (naked DNA), contendo o gene do hormônio de crescimento humano (hGH), no músculo quadríceps de camundongos anões (lit/lit) e anões imunodeficientes (lit/scid). Foram padronizadas as condições de eletroporação em 8 pulsos de 50 V e 20 ms com 0,5 s de intervalo, utilizando um plasmídeo com o promotor da ubiquitina C e a sequência genômica do hGH. A administração de uma dose única de 50 g do plasmídeo pUC-UBI-hGH em camundongos lit/scid, seguida de eletroporação, propiciou pela primeira vez na literatura obtenção de níveis sustentáveis na circulação de 1,5-3,0 ng hGH/ml, durante 60 dias. Esses animais tratados com DNA apresentaram um aumento de peso altamente significativo (P<0,001) de 33,1%, comparado a uma perda de peso, não significativa, no grupo controle (camundongos injetados com salina + eletroporação). Os músculos quadríceps dos animais tratados apresentaram um aumento de 48%, quando comparados aos dos animais do grupo controle (P<0,001). Outro estudo de eletrotransferência foi realizado comparando a utilização da sequência genômica do hGH (gDNA) com a complementar (cDNA), em plasmídeos sob o controle do promotor de citomegalovírus (CMV). Foi observado que o cDNA do hGH foi expresso de maneira mais eficiente na circulação de camundongos lit/scid, por um período de 21 dias. Foram também construídos vetores lentivirais com os genes do hGH (cDNA e gDNA) e do mGH (cDNA), que serão utilizados num próximo estudo, tanto para a transdução de queratinócitos, como para a eletrotransferência in vivo. Vetores lentivirais serão de fato necessários para futuros estudos devido a sua reduzida toxicidade em comparação com os retrovirais. Os resultados deste trabalho, assim como as perspectivas de estudo abertas, têm como meta estabelecer condições para que a terapia gênica seja num futuro próximo uma alternativa viável e segura para o tratamento da deficiência de GH e de outras doenças sistêmicas. / Keratinocytes are very attractive cells for ex vivo gene transfer and systemic delivery, since proteins secreted by these cells may reach the circulation via a mechanism which mimics the natural process. In this study, retrovirally transduced keratinocytes with the mouse growth hormone (mGH) gene underwent a treatment for adhesion to collagen and clonal analysis, in order to enrich in stem cells the keratinocyte population. The main result was an in vitro increase of cell viability for treated keratinocytes, which should result in an increase of the in vivo sustainability of hormone secretion, when implanting organotypic cultures onto immunodeficient dwarf (lit/scid) mice. A model for in vivo gene therapy based on the electrotransfer of human growth hormone (hGH)-coding naked DNA in the exposed quadriceps muscle of dwarf (lit/lit) and immunodeficient dwarf (lit/scid) mice was also utilized. Electroporation conditions were optimized, setting up eight 50-V pulses of 20 ms at a 0.5 s interval, using a plasmid containing the ubiquitin C promoter and the genomic hGH sequence. Administration of a single dose of 50 g of this plasmid led, for the first time in the literature, to sustained levels of circulating hGH of the order of 1.5-3 ng/ml, up to 60 days. The DNA-treated animals presented a highly significant weight gain (P<0.001) of 33.1%, compared to a non-significant weight loss of 4.2% for the control group (saline injected mice + electroporation). The injected quadriceps muscles of the DNA-treated mice showed a 48% weight increase, when compared to the control group (P<0.001). Another electrotransfer study was carried out comparing the use of the genomic hGH sequence (gDNA) with the complementary sequence (cDNA), cloned under the control of the cytomegalovirus (CMV) promoter. It was observed that hGH was more efficiently expressed in the circulation of lit/scid mice, for 21 days, when injecting cDNA. Lentiviral vectors containing the hGH (cDNA and gDNA) and the mGH (cDNA) genes were also constructed and will be used in a next study, both for the transduction of keratinocytes or for in vivo electrotransfer. Lentiviral vectors will in fact be necessary for future studies, considering their reduced toxicity when compared to retroviral vectors. The results of this work, as well as opening perspectives of new studies, will establish in the near future conditions for gene therapy as a feasible and safe option for the treatment of GH deficiency and other systemic diseases.

Queratinócitos são células bastante atrativas para a transferência gênica ex vivo e liberação sistêmica, uma vez que as proteínas secretadas por estas células podem atingir a circulação via um mecanismo similar ao processo natural. No presente trabalho, queratinócitos transduzidos mediante um vetor retroviral com o gene do hormônio de crescimento de camundongo (mGH) foram submetidos a um tratamento de aderência ao colágeno e à análise clonal, com o intuito de enriquecer esta população de queratinócitos em células-tronco. O principal resultado foi um aumento da viabilidade celular in vitro dos queratinócitos tratados, que poderá se refletir num aumento da durabilidade da secreção do hormônio in vivo, quando realizado o implante de culturas organotípicas em camundongos anões imunodeficientes (lit/scid). Foi também utilizado um modelo de terapia gênica in vivo, baseado na eletrotransferência de DNA plasmidial (naked DNA), contendo o gene do hormônio de crescimento humano (hGH), no músculo quadríceps de camundongos anões (lit/lit) e anões imunodeficientes (lit/scid). Foram padronizadas as condições de eletroporação em 8 pulsos de 50 V e 20 ms com 0,5 s de intervalo, utilizando um plasmídeo com o promotor da ubiquitina C e a sequência genômica do hGH. A administração de uma dose única de 50 g do plasmídeo pUC-UBI-hGH em camundongos lit/scid, seguida de eletroporação, propiciou pela primeira vez na literatura obtenção de níveis sustentáveis na circulação de 1,5-3,0 ng hGH/ml, durante 60 dias. Esses animais tratados com DNA apresentaram um aumento de peso altamente significativo (P<0,001) de 33,1%, comparado a uma perda de peso, não significativa, no grupo controle (camundongos injetados com salina + eletroporação). Os músculos quadríceps dos animais tratados apresentaram um aumento de 48%, quando comparados aos dos animais do grupo controle (P<0,001). Outro estudo de eletrotransferência foi realizado comparando a utilização da sequência genômica do hGH (gDNA) com a complementar (cDNA), em plasmídeos sob o controle do promotor de citomegalovírus (CMV). Foi observado que o cDNA do hGH foi expresso de maneira mais eficiente na circulação de camundongos lit/scid, por um período de 21 dias. Foram também construídos vetores lentivirais com os genes do hGH (cDNA e gDNA) e do mGH (cDNA), que serão utilizados num próximo estudo, tanto para a transdução de queratinócitos, como para a eletrotransferência in vivo. Vetores lentivirais serão de fato necessários para futuros estudos devido a sua reduzida toxicidade em comparação com os retrovirais. Os resultados deste trabalho, assim como as perspectivas de estudo abertas, têm como meta estabelecer condições para que a terapia gênica seja num futuro próximo uma alternativa viável e segura para o tratamento da deficiência de GH e de outras doenças sistêmicas. / Keratinocytes are very attractive cells for ex vivo gene transfer and systemic delivery, since proteins secreted by these cells may reach the circulation via a mechanism which mimics the natural process. In this study, retrovirally transduced keratinocytes with the mouse growth hormone (mGH) gene underwent a treatment for adhesion to collagen and clonal analysis, in order to enrich in stem cells the keratinocyte population. The main result was an in vitro increase of cell viability for treated keratinocytes, which should result in an increase of the in vivo sustainability of hormone secretion, when implanting organotypic cultures onto immunodeficient dwarf (lit/scid) mice. A model for in vivo gene therapy based on the electrotransfer of human growth hormone (hGH)-coding naked DNA in the exposed quadriceps muscle of dwarf (lit/lit) and immunodeficient dwarf (lit/scid) mice was also utilized. Electroporation conditions were optimized, setting up eight 50-V pulses of 20 ms at a 0.5 s interval, using a plasmid containing the ubiquitin C promoter and the genomic hGH sequence. Administration of a single dose of 50 g of this plasmid led, for the first time in the literature, to sustained levels of circulating hGH of the order of 1.5-3 ng/ml, up to 60 days. The DNA-treated animals presented a highly significant weight gain (P<0.001) of 33.1%, compared to a non-significant weight loss of 4.2% for the control group (saline injected mice + electroporation). The injected quadriceps muscles of the DNA-treated mice showed a 48% weight increase, when compared to the control group (P<0.001). Another electrotransfer study was carried out comparing the use of the genomic hGH sequence (gDNA) with the complementary sequence (cDNA), cloned under the control of the cytomegalovirus (CMV) promoter. It was observed that hGH was more efficiently expressed in the circulation of lit/scid mice, for 21 days, when injecting cDNA. Lentiviral vectors containing the hGH (cDNA and gDNA) and the mGH (cDNA) genes were also constructed and will be used in a next study, both for the transduction of keratinocytes or for in vivo electrotransfer. Lentiviral vectors will in fact be necessary for future studies, considering their reduced toxicity when compared to retroviral vectors. The results of this work, as well as opening perspectives of new studies, will establish in the near future conditions for gene therapy as a feasible and safe option for the treatment of GH deficiency and other systemic diseases.

Magister Scientiae Dentium - MSc(Dent) / The Human Immunodeficient Virus (HIV) is escalating in South Africa at an alarming rate. The impact of HIV today and in the future could have grave consequences for the South African population as it affects adults in their most productive years. To ease costs on the health system, health workers should be familiar with HIV patients needs in general, and specifically in areas such as oral disease which can contribute to the wellness or ill health of the patient. This could facilitate more appropriate and cost effective care ofHIV patients. World-wide reports indicate that the HIV virus is more prevalent in females than males. Women are also experiencing greater virulence of HIV and therefore greater severity of the disease. This research assessed whether there were differences in the prevalence and severity of oral symptoms ofHIV positive men and women. Oral health practices were also examined. As oral disease is very prevalent in HIV positive persons and has been a neglected area for research and program development, it was included in this study. This study also aimed to assess the perceived needs of patients affected by HIV. Such a study presents HIV positive patients an opportunity to participate in a process which allows patients to voice their needs and problems, as well as be involved in setting priorities. The study sought to assess whether needs differed according to the patients gender, age and symptom levels. A needs questionnaire with five domains which included medical and oral needs, social, economic, psychological and informational domains of needs was developed. The measure also included a section on demographics and oral health questions, and was administered as a structured interview. The sample consisted of 338 HIV positive males and females residing in Cape Town and its environs and attending the Out Patients' Departments of three major provincial hospitals, as well as two community clinics during May to November of 1995.